Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
Specific mutations in amino acid sequence are represented herein as “Xaa1nXaa2” where Xaa1 is the original amino acid residue before mutation, n is the residue number and Xaa2 is the mutant amino acid. The abbreviation “Xaa” may be the three letter or single letter amino acid code. A mutation in single letter code is represented, for example, by X1nX2 where X1 and X2 are the same as Xaa1 and Xaa2, respectively. The amino acid residues for Hepatitis B virus DNA polymerase are numbered with the residue methionine in the motif “Tyr Met Asp Asp (YMDD),” being residue number 550, wich corresponds to residue number 159 of SEQ ID NO:8.
The reference HBV is considered herein to comprise a composite or consensus nucleotide or amino acid sequence from HBV genotypes A through G (1, 2).
The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating advances in the medical and allied health fields. This is particularly the case with the generation of therapeutic compositions and recombinant vaccines. Recombinant technology is providing the genetic bases for screening or identifying useful components for therapeutic compositions.
Hepatitis B virus (HBV) can cause debilitating disease conditions ranging from subclinical infection to chronic active hepatitis and can lead to acute liver failure or fulminant hepatitis.
Most patients will suffer an acute hepatitis during which time the virus is eliminated. In fulminant hepatitis, patients have acute liver failure and this frequently leads to patient death. About 5% of patients in North America and Europe fail to eliminate the virus, whereas in West Africa, up to 15% of infected patients fail to clear HBV (3). Persistent HBV infection predisposes the host to chronic liver disease and hepatocellular carcinoma (4).
The HBV genome comprises a series of overlapping genes in a circular, partially double-standard DNA molecule (5) [see also FIG. 1]. These genes encode for four overlapping open reading frames. For example, the gene encoding the DNA polymerase overlaps the viral envelope genes (Pre-S1, Pre-S2 and S) and partially overlaps the X and core genes. The protein component of the small HBV surface protein is generally referred to as the HBV surface antigen (HBsAg) and is encoded by the S gene sequence. The Pre-S1 and Pre-S2 gene sequences encode the other envelope components (6). The core open reading frame encodes for both the hepatitis B core protein (HBcAg) and HBeAg, which starts from a precore initiation codon. HBV variants can have single or multiple mutations in one or more of the overlapping genes.
The HBV DNA polymerase is a reverse transciptase (i.e. an RNA dependent DNA polymerase) and also has DNA dependent DNA polymerase as well as primase and RNase H activity. Nucleoside analogues have been used to inhibit HBV DNA replication. However, mutations have arisen in the gene encoding the HBV DNA polymerase resulting in the development of HBV variants resistant to the nucleoside analogues. Resistance may occur to a single nucleoside analogue or cross-resistance may also occur to an entire family of nucleoside analogues. Furthermore, when the mutation occurs in a region overlapping with the gene encoding HBsAg, alterations may occur to the HBsAg itself leading to the development of vaccine escape mutants.
Some precore variants of HBV result in hepatitis B e antigen (HBeAg)-negative hepatitis B. Seven to 30% of patients with chronic HBV infection worldwide are HBeAg-negative and are positive for HBV DNA by hybridisation using commercial tests. One such variant is unable to synthesize HBeAg. A single base substitution (G-to-A) at nucleotide 1896 (A1896; numbering from the unique EcoRI site) gives rise to a translational stop codon in the second last codon (codon 28) of the precore gene. Other precore and basal core promoter (BCP) mutations are listed in Table 1. Since the core gene itself is not affected, synthesis of the core protein proceeds normally enabling production of virions. Precore A1896 mutations occur in both anti-HBe-positive patients with mild disease and those with high level viraemia and severe chronic hepatitis, suggesting that there is not a direct causal association with chronic progressive disease. However, infection with precore mutant virus has been associated with fulminant hepatitis and in the transplantation setting, graft failure (15).
The HBsAg comprises an antigenic region referred to as the “a” determinant (7). The “a” determinant is complex, conformational and dependent upon disulphide bonding among highly conserved cysteine residues. Genetic variation leading to changes in the “a” determinant has been implicated in mutants of HBV which escape the immunological response generated to conventional vaccines (8-12). One particularly common mutation is a glycine (G) to arginine (R) substitution at amino acid position 145 (G145R) of HBsAg. This mutation affects the “a” epitope region.
The increasing reliance on chemical and immunological intervention in treating or preventing HBV infection is resulting in greater selective pressure for the emergence of variants of HBV which are resistant to the interventionist therapy. Due to the overlapping genomic structure of HBV, HBV variants, may be directly or indirectly selected for by the use of chemical agents or vaccines.
It is important to be able to detect variant HBVs so that appropriate steps can be taken to modify a therapeutic protocol. This is also particularly important in the development of new therapeutic agents to be effective against known resistant variants of HBV and also when cross resistance develops within a family of chemically related anti-viral agents.
HBV baculovirus mediated HBV replication is a transient system and does not require integration of the HBV viral genome. This system was recently described by Delaney et al. (13, 14). The HBV baculovirus system has a number of advantages over standard transient transfection systems and cell lines expressing HBV.
In the study of HBV replication and the development of therapeutic agents directed against HBV, some cell lines have been developed which arc capable of expressing HBV DNA. However, these cell lines were developed using HBV DNA sequences under the control of heterologous promoters or heterologous regulatory sequences which are unlikely to mimic the situation in a naturally infected cell.
Furthermore, cell lines commonly used to study HBV contain multiple copies of integrated HBV DNA. Hepadnavirus genomes are maintained in the nucleus of infected cells in vivo as a pool of episomal, covalently closed circular (CCC) DNA molecules. Although the integration of HBV DNA in human liver has been reported, it is not an obligatory part of the HBV lifecycle and integration is not required for HBV replication. In addition, when integrated HBV DNA is found, it is frequently rearranged and is often transcriptionally silent. Because HBV expressing cell lines contain stably integrated HBV DNA, viral gene expression and replication is continuous; therefore, it is not possible to experimentally control the time or conditions under which these processes are initiated. Stable HBV expressing cell lines contain fixed numbers of integrated typically head-to tail orientated HBV genomes and, as such, HBV gene expression and replication levels cannot be regulated and are restricted to the number of integrated copies which each cell line contains. Consequently, it is not possible to study the effects of increasing or decreasing the copy number of integrated HBV genomes without transfecting the cell line and/or selecting new cell lines.
HBV baculovirus infection, even at high multiplicities, is not toxic to cells such as HepG2 or Huh-7. HBV expression can be enhanced or prolonged in a population of HBV baculovirus infected cells simply by superinfection of the cultures.
One major difference between baculovirus-mediated gene transfer of HBV to HepG2 cells and stably transfected cell lines is the ability to synchronously initiate the replication process. In a stably transfected HBV cell line, such as a derivative of the HepG2 cell line referred to as “2.2.15”, each cell contains virus at all phases of the replication cycle. In contrast, HBV baculovirus infection can be used to synchronously start HBV replication in, for example, HepG2 cells because these cells contain no viral products before infection. In HBV baculovirus infected HepG2 cells, it is possible to follow the time course for secretion of both HBsAg and HBeAg with time after infection using the appropriate recombinant HBV baculovirus.
There is a need, therefore, to develop a baculovirus system to screen for specific HBV variants having altered sensitivities to particular agents.